Method for isolating osteopontin using concentrated feeds

ABSTRACT

The present invention pertains to a method for isolating osteopontin from milk-derived feeds having a high concentration of protein. Particularly, the present method involves the use of a narrow window of pH and specific conductance of the milk-derived feed, which surprisingly has proven to provide a very efficient isolation of osteopontin from chemically complex feeds.

The present Application is a U.S. National Phase ofPCT/EP2012/053749filed on Mar. 5, 2012 (“PCT Application”), which claimspriority from U.S. Provisional Application No. 61/448,775 filed on Mar.3, 2011 and European Patent Application No. EP 11156826.7, filed on Mar.3, 2011. The PCT Application, the U.S. Provisional application and theEP Patent Application are hereby incorporated by reference in theirentirety into the present Application. The PCT application, incorporatedby reference herein, includes any amendments entered in the PCTapplication.

FIELD OF THE INVENTION

The present invention pertains to a method for isolating osteopontinfrom a milk-derived feed, e.g. a feed based on milk serum, sweet whey,or acid whey. Particularly, the present method involves the use of arelatively high protein concentration and a narrow window of pH andspecific conductance of the milk-derived feed, which surprisingly hasproven to provide a very efficient isolation of osteopontin fromchemically complex feeds.

BACKGROUND

Osteopontin is an acidic, highly phosphorylated, sialic acid rich,calcium binding protein. Osteopontin contains approx. 28 moles of boundphosphate per mol osteopontin and binds approx. 50 moles of Ca per moleosteopontin.

Osteopontin (OPN) is a multifunctional bioactive protein that isimplicated in numerous biological processes, such as bone remodelling,inhibition of ectopic calcification, and cellular adhesion andmigration, as well as several immune functions. Osteopontin hascytokine-like properties and is a key factor in the initiation of Thelper 1 immune responses. Osteopontin is present in most tissues andbody fluids, with the highest concentrations being found in milk.

Supporting an inhibitory function of OPN in ectopic calcification, an invivo model using OPN-deficient mice showed diminished calcification uponexogenous addition of the protein. In addition, OPN is involved in theurinary tract's defence against the formation of renal stones becauseOPN can inhibit growth and aggregation of calcium oxalate monohydratecrystals.

The biological role of OPN in milk is not clear; however, severalfunctions could be hypothesized. Osteopontin has been reported to beinvolved in mammary gland development and differentiation, and highlevels of OPN expression have been observed in the mammary gland inearly lactation. Furthermore, the highly anionic nature of the proteincould enable OPN to form soluble complexes with calcium ions and therebyinhibit unintentional calcium crystallization and precipitation in milk.

In the scientific literature osteopontin is typically purified from boneor milk and it is typically present in bovine milk in a concentration of20 mg/L. In milk, osteopontin is a serum protein but may also to someextent associate with the casein micelles depending on the Ca²⁺ level.Acid whey is the preferred raw material for industrial production ofosteopontin. When acid whey is formed osteopontin is thought to leavethe casein micelles as Ca²⁺ leaks out into the serum phase. This aspectmakes acid whey a straightforward source of osteopontin. For the samereason sweet whey has a slightly lower osteopontin content. Furthermore,sweet whey contains caseino macropeptide (CMP) from enzymatic cleavageof the kappa-casein. CMP has many biochemical resemblances withosteopontin—both are small, flexible, acidic, phosphorylatedglycoproteins. For this reason CMP and osteopontin is believed to bequite similar in their binding to ion exchange resins, which will pose aproblem in purifying osteopontin from a CMP-containing raw material.Another aspect is the likely degradation of osteopontin by proteolyticenzymes used for cheese making. These three aspects may have resulted inavoidance of this raw material for osteopontin purification, both forindustrial production and for scientific research.

PRIOR ART

WO 02/28413 A1 describes a method of producing an osteopontin-containingcomposition from feedstocks, such as milk and acid whey, by means of lowpH anion exchange. In WO 02/28413 A1 it is emphasised that neither theprocess feedstock nor the resulting product may contain cGMP, which is atype of CMP which is known to bind to anion exchangers and which wouldinhibit the binding of osteopontin.

WO 01/149741 A2 describes a process wherein osteopontin is purified froma milk material by mixing the milk material with soluble calcium andadjusting the pH of the mixture to selectively precipitate the otherprotein components of whey while keeping osteopontin in solution.

SUMMARY OF THE INVENTION

The present inventors have discovered that, surprisingly and contrary tothe prejudices found in the prior art, osteopontin can be isolated byanion exchange in high yield and high purity from complex milk-derivedfeeds despite the presence of competing proteins of the feed. Thepresent inventors have found that the specific conductance of the feedis particularly important for the yield and purity of osteopontin andhave identified an optimum window of the specific conductance for theisolation of osteopontin from milk derived feeds.

Thus, an aspect of the invention relates to a method for isolatingosteopontin from a milk-derived feed, the method comprising the stepsof:

-   -   a) providing a milk-derived feed comprising osteopontin, said        milk-derived feed having a pH in the range of pH 3.6-6.5 at 25        degrees C. and a specific conductance in the range of 4-10 mS/cm        at 25 degrees C.,    -   b) subjecting said milk-derived feed to anion exchange        chromatography, which includes contacting the milk-derived feed        with an anion exchange medium,    -   c) optionally, washing the anion exchange medium, and    -   d) recovering the protein bound to the anion exchange medium,        thereby obtaining a composition comprising isolated osteopontin.

An aspect of the invention may for example relate to a method forisolating osteopontin from a milk-derived feed, the method comprisingthe steps of:

-   -   a) providing a milk-derived feed comprising osteopontin, said        milk-derived feed having a pH in the range of pH 3.6-6.5 at 25        degrees C. and a specific conductance in the range of 4-10 mS/cm        at 25 degrees C., and wherein the milk-derived feed comprises a        total amount of protein in the range of 50-250 g/L milk-derived        feed,    -   b) subjecting said milk-derived feed to anion exchange        chromatography, which includes contacting the milk-derived feed        with an anion exchange medium,    -   c) optionally, washing the anion exchange medium, and    -   d) recovering the protein bound to the anion exchange medium,        thereby obtaining a composition comprising isolated osteopontin.

In addition to the above-mentioned advantages, the present method allowsfor a more cost-efficient use of the anion exchange medium, and a higheryield of osteopontin per kg anion exchange medium.

Another advantage of the present method is an improved yield ofosteopontin per anion exchange cycle as demonstrated in Example 5.

The “specific conductance” (sometimes referred to as the “specificconductivity”) of an aqueous solution is a measure of the ability of thesolution to conduct electricity. The specific conductance may e.g. bedetermined by measuring the AC resistance of the solution between twoelectrodes and the result is typically given in the unit miliSiemens percm (mS/cm). The measurement of specific conductance may for example bemeasured according to the EPA (the US Environmental Protection Agency)Method No. 120.1.

Yet an aspect of the invention relates to the osteopontin-containingcomposition obtained by the present method.

A further aspect of the invention pertains to an osteopontin-containingcomposition as such.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The effect of pH of sweet whey-derived feed on purity and yieldof osteopontin at a specific conductance of 5.5 mS/cm,

FIG. 2. The effect of specific conductance of sweet whey-derived feed onpurity and yield of osteopontin at pH 4.3, and

FIG. 3. The influence of specific conductance of acid whey derived feedon OPN purity and yield at pH 4.3.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned, an aspect of the invention pertains to a method forisolating osteopontin from a milk-derived feed, the method comprisingthe steps of:

-   -   a) providing a milk-derived feed comprising osteopontin, said        milk-derived feed having a pH in the range of pH 3.6-6.5 at 25        degrees C. and a specific conductance in the range of 4-10 mS/cm        at 25 degrees C., and the wherein milk-derived feed comprises a        total amount of protein in the range of 50-250 g/L milk-derived        feed,    -   b) subjecting said milk-derived feed to anion exchange        chromatography, which includes contacting the milk-derived feed        with an anion exchange medium,    -   c) optionally, washing the anion exchange medium, and    -   d) recovering the protein bound to the anion exchange medium,        thereby obtaining a composition comprising isolated osteopontin.

The steps of the method are typically performed in sequence, e.g. stepsa), b), c) and d). However, in some embodiments of the invention step c)of the method is omitted and in this case the method comprises the stepsa), b), and d).

In the context of the present invention the term “isolating osteopontin”relates to enrichment of osteopontin to a weight percent of at least 70%(w/w), preferably at least 80% (w/w), and even more preferably at least90% (w/w) relative to the total weight of protein recovered from theanion exchange medium during step d). Isolating osteopontin may forexample involve the enrichment of osteopontin to a weight percent of atleast 95% (w/w), such as at least 97% (w/w) relative to the total weightof protein recovered from the anion exchange medium during step d). Themethod is particularly useful for improving the selective enrichment ofosteopontin from a milk-derived feed comprising additional proteinhaving an isoelectric point (pI) <5.0, for example milk-derived feedswhich contain CMP. The term “selective enrichment” should be understoodas increasing the molar ratio between osteopontin and the total amountof other proteins of the milk-derived feed.

The isoelectric point of a protein is preferably determined byisoelectric focusing at 25 degrees C. In the context of the presentinvention the term “milk-derived feed” pertains to the liquid feed whichcontacts the anion exchange medium.

The milk-derived feed is derived from milk from one or more mammaliansource(s), e.g. milk from human, cow, sheep, goat, buffalo, camel,llama, horse and/or deer. In some preferred embodiments of the inventionthe milk-derived feed is derived from bovine milk.

In the context of the present invention, the terms “milk-derived feed”,“sweet whey-derived feed”, “acid whey-derived feed”, and “milkserum-derived feed” relate to feeds wherein at least 50% (w/w) of thetotal protein originates from milk, sweet whey, acid whey or milk serum,respectively. In some preferred embodiments of the invention at least90% (w/w), and preferably substantially all, of the total protein of themilk-derived feed originates from a milk, sweet whey, acid whey or milkserum.

In the context of the present invention, the term “milk” relates to theliquid obtained from the mammary glands of mammals during lactation. Theterm “milk” should be interpreted broadly and covers both the raw milk,i.e. the liquid obtained directly from the mammary glands, andstandardised milk products such as e.g. skimmed milk or whole milk,where the concentration of the milk fat has been reduced relative to theoriginal raw milk.

Whey is a collective term referring to the watery by-product which isproduced during the manufacture of cheese or casein from milk.

In the context of the present invention, the term “sweet whey” relatesto whey, which is obtained during rennet-based coagulation of milk,which for example takes place during the production of yellow cheese.

In the context of the present invention, the term “acid whey” relates towhey, which is obtained during chemical or biological acidification ofmilk, which for example takes place during the production of cottagecheese or quark, or in the production of casein/caseinates.

In the context of the present invention, the term “milk serum”, whichalso is known as “serum whey”, “native whey” or “lactoserum”, pertainsto milk from which milk fat and casein micelles have been removed.However, milk serum typically contains some free casein species, whichhave dissociated from the native casein micelles before the micelleswere removed. Milk serum may for example be produced by microfiltering askimmed milk through a filter or membrane having a pore size of approx.0.1 micrometer and collecting the resulting permeate as the milk serum.Milk serum may be produced according to Evans et al.

As will be understood, the milk, sweet whey, acid whey or milk serum mayhave been subjected to several process steps to prepare the milk-derivedfeed.

Processing of milk or milk-related products typically involves one ormore heat treatment processes, such as a pasteurisation (e.g. 72 degreesC. for 15 sec) or a high pasteurisation (e.g. 85 degrees C. for 20 sec).

Alternatively, or additionally, the processing may involve one or morefiltration step(s). Microfiltration may be used to removemicroorganisms, or alternatively to concentrate casein. Ultrafiltrationor nanofiltration may e.g. be used to concentrate whey protein or milkserum protein.

Alternatively, or additionally, the processing may involve one or morecentrifugation step(s), e.g. for separating fat from skimmed milk and/orseparating microorganisms from the milk.

Alternatively, or additionally, the processing may involve one or moreevaporation step(s) for removing water and thus concentrating dry mattersuch at proteins and/or minerals.

The processing may also comprise one or more pH adjustment(s).Acidification may e.g. be used to coagulate casein and pH adjustment mayfurthermore be important when the processing involves ion exchangechromatography.

In some embodiments of the invention the milk-derived feed comprises onemore process stream(s) from the production of other milk proteinfractions, such as filtered, heat treated whey or alpha-lactalbumin-and/or beta-lactoglobulin-depleted whey.

In some preferred embodiments of the invention the milk-derived feedcomprises, or even essentially consists of, a milk serum-derived feed.The milk-derived feed may for example be a milk serum, and preferably amilk serum protein concentrate, e.g. in the form of the retentateobtained by ultrafiltration of milk serum.

The content of osteopontin in the milk-derived feed depends on thespecific feed type. In some embodiments of the invention themilk-derived feed comprises osteopontin in an amount in the range of0.01-20% (w/w) relative to the total amount of protein of themilk-derived feed. For example, the milk-derived feed may compriseosteopontin in an amount in the range of 0.05-5% (w/w) relative to thetotal amount of protein of the milk-derived feed. The milk-derived feedmay e.g. comprise osteopontin in an amount in the range of 0.1-2% (w/w).

Alternatively, the milk-derived feed may comprise osteopontin in anamount in the range of 0.1-1% (w/w) relative to the total amount ofprotein of the milk-derived feed.

In the context of the present invention, a product, component, method,or method step which is stated to “essentially consist of one or moresub-components or one or more activities consists of the one or morespecifically mentioned sub-components or the one or more specificallymentioned activities but may also include one or more additional unnamedsub-components or activities which do not materially affect the basicand novel characteristic(s) of the present invention.

As mentioned above, the present method is particularly effective forisolating osteopontin from complex feeds, i.e. feeds which containmolecular entities that interfere with the isolation of osteopontin,such as proteins having a pI lower than 5.0. Such proteins have beenfound to compete with osteopontin for the functional groups of the anionexchange medium.

Examples of proteins having a pI<5.0 are alpha lactalbumin, proteosepeptone-3, proteose peptone-5, and proteose peptone-8, andcasein-derived peptides such as caseino macropeptide and/or caseinophosphopeptides. Thus the additional protein may comprise one or moreproteins from the group consisting of alpha lactalbumin, proteosepeptone-3, proteose peptone-5, and proteose peptone-8, casein-derivedpeptide, caseino macropeptide, caseino phosphopeptide, and combinationsthereof. Free alpha-casein and free beta-casein are other example ofproteins having a pI value <5.0.

In some embodiments of the invention the milk-derived feed comprises anamount of additional protein having a pI<5.0 of at least 0.1% (w/w)relative to the total amount of protein of the milk-derived feed. Forexample, the milk-derived feed may comprise an amount of additionalprotein having a pI<5.0 of at least 0.5% (w/w) relative to the totalamount of protein of the milk-derived feed.

Alternatively, the milk-derived feed may comprise an amount ofadditional protein having a pI<5.0 of at least 2% (w/w) relative to thetotal amount of protein of the milk-derived feed. As will be understood,the additional protein having a pI<5.0 does not include osteopontin buttypically includes one or more other proteins having a pI in thespecified range.

In other embodiments of the invention the milk-derived feed comprise anamount of additional protein having a pI<5.0 in the range of 0.1-50%(w/w) relative to the total amount of protein of the milk-derived feed.For example, the milk-derived feed may comprise an amount of additionalprotein having a pI<5.0 in the range of 0.5-40% (w/w) relative to thetotal amount of protein of the milk-derived feed. Alternatively, themilk-derived feed may comprise an amount of additional protein having apI<5.0 in the range of 2-25% (w/w) relative to the total amount ofprotein of the milk-derived feed.

The milk-derived feed may e.g. comprise an amount of additional proteinhaving a pI<5.0 in the range of 0.1-20% (w/w) relative to the totalamount of protein of the milk-derived feed. For example, themilk-derived feed may comprise an amount of additional protein having apI<5.0 in the range of 0.3-15% (w/w) relative to the total amount ofprotein of the milk-derived feed. Alternatively, the milk-derived feedmay comprise an amount of additional protein having a pI<5.0 in therange of 0.5-10% (w/w) relative to the total amount of protein of themilk-derived feed. In further embodiments of the invention themilk-derived feed comprises an amount of additional protein having apI<5.0 in the range of 1-50% (w/w) relative to the total amount ofprotein of the milk-derived feed. For example, the milk-derived feed maycomprise an amount of additional protein having a pI<5.0 in the range of10-45% (w/w) relative to the total amount of protein of the milk-derivedfeed. Alternatively, the milk-derived feed may comprise an amount ofadditional protein having a pI<5.0 in the range of 15-40% (w/w) relativeto the total amount of protein of the milk-derived feed.

In some preferred embodiments of the invention, the additional proteinhaving a pI<5.0 has a pI<4.5, and preferably a pI<4.0.

In the context of the present invention the term “protein” encompassesboth large aggregates of polypeptides, single polypeptide chains andpeptides such as di- or tri-peptides. Chemically, proteins are polymerscomprising different and/or identical amino acids linked by so-calledpeptide bonds.

In some preferred embodiments of the invention the milk-derived feedfurthermore comprises caseino macropeptide (CMP).

In the context of the present invention, the term “CMP” or “caseinomacropeptide” pertains to a small protein, which is released fromkappa-casein upon exposure to rennet enzymes. CMP encompasses bothglycosylated variants and a non-glycosylated variant of the protein. Theglycosylated variants of the protein is sometimes referred to as caseinoglycomacropeptide (cGMP).

In other preferred embodiments of the invention the milk-derived feedcomprises, or even essentially consists of, sweet whey-derived feed. Themilk-derived feed may for example be sweet whey, and preferably sweetwhey protein concentrate, e.g. in the form of the retentate obtained byultrafiltration of sweet whey.

In some embodiments of the invention the milk-derived feed may comprise,or even essentially consists of, an acid whey-derived feed. Themilk-derived feed may for example be an acid whey, and preferably anacid whey protein concentrate, e.g. in the form of the re entateobtained by ultrafiltration of acid whey.

In the context of the present invention, the terms “milk serum proteinconcentrate”, “sweet whey protein concentrate”, or “acid whey proteinconcentrate” pertains to an aqueous composition which contains at least80% (w/w) of the total protein which was present in original milk serum,sweet whey, or acid whey, respectively, and which has a total proteincontent of at least 25% (w/w) relative to the dry weight of the aqueouscomposition.

However, in other embodiments of the invention the milk-derived feed isnot an acid whey-derived feed.

In preferred embodiments of the invention the milk-derived feed, e.g. asweet whey-derived feed, comprises CMP in an amount of at least 1% (w/w)relative to the total amount of protein of the milk-derived feed. Forexample, the milk-derived feed, e.g. a sweet whey-derived feed, maycomprise CMP in an amount of at least 5% (w/w) relative to the totalamount of protein of the milk-derived feed, preferably at least 10%(w/w), and even more preferably at least 15% (w/w) relative to the totalamount of protein of the milk-derived feed.

In some embodiments of the invention the milk-derived feed, e.g. a sweetwhey-derived feed, comprises CMP in an amount in the range of 1-40%(w/w) relative to the total amount of protein of the milk-derived feed.For example, the milk-derived feed, e.g. a sweet whey-derived feed, maycomprise CMP in an amount in the range of 5-35% (w/w) relative to thetotal amount of protein of the milk-derived feed, preferably in therange of 10-30% (w/w), and even more preferably in the range of 15-25%(w/w) relative to the total amount of protein of the milk-derived feed.

The present inventors have observed that, surprisingly, a precipitate isformed during the present process when the feed is based on milk serumor a concentrate thereof. The precipitate causes problems during theanion exchange process and reduces its robustness.

The inventors have investigated the precipitate and found indicationsthat it contains precipitated casein species, which were present indissolved form such as free beta-casein or free alpha-casein.

In the context of the present invention, the terms “free beta-casein” or“free alpha-casein” pertains beta-casein molecules or alpha-caseinmolecules which are not bound to the native casein micelles of milkproducts. Such free beta-casein or free alpha-casein includes dissolvedsingle molecules of beta-casein or alpha-casein or small aggregates ofalpha-casein and/or beta-casein. Single molecules of beta-casein are forexample known to form small beta-casein micelles, which also is anexample of free beta-casein.

Thus, in some preferred embodiments of the invention the milk-derivedfeed furthermore comprises free alpha-casein and free beta-casein. Freealpha-casein and free beta-casein are typically present in milk-serumderived feed.

The inventors have found that this precipitation problem can be solvedby removing the precipitate from the milk derived feeds prior to anionexchange. Thus, in some preferred embodiments of the invention step a)of the method involves removing precipitate from the acidified liquidwhich is to form the milk-derived feed. Such removal may e.g. involvecentrifugation or filtration of the acidified liquid.

Another solution to the precipitate problem is to use a pH of the feedwhere precipitation is limited or even absent, e.g. in the range of pH5.0-6.5.

The milk-derived feed may therefore have a pH in the range of 5.0-6.5,and preferably in the range of pH 5.0-6.0.

When performing the anion exchange in this pH range it is furthermoresuggested to use a feed/process temperature above 15 degrees C., such as20-40 degrees C. In this temperature range dissolved beta-casein formssmall beta-casein micelles, not to be confused with native caseinmicelles of milk, and these beta-casein micelles seem to interfere lesswith the anion exchange process than single beta-casein molecules.

The temperature of the milk-derived feed and anion exchange materialduring step b) may therefore be in the range of 15-40 degrees C., andpreferably in the range of 20-38 degrees C.

The milk-derived feed may e.g. comprise a total amount of freealpha-casein and free beta-casein of at least 0.5% (w/w) relative to thetotal amount of protein of the milk-derived feed. For example, themilk-derived feed may comprise a total amount of free alpha-casein andfree beta-casein of at least 2% (w/w) relative to the total amount ofprotein of the milk-derived feed. The milk-derived feed may e.g.comprise a total amount of free alpha-casein and free beta-casein of atleast 10% (w/w) relative to the total amount of protein of themilk-derived feed.

In some embodiments of the invention the milk-derived feed comprises atotal amount of free alpha-casein and free beta-casein in an amount inthe range of 0.5-40% (w/w) relative to the total amount of protein ofthe milk-derived feed. For example, the milk-derived feed may comprise atotal amount of free alpha-casein and free beta-casein in an amount inthe range of 2-20% (w/w) relative to the total amount of protein of themilk-derived feed. The milk-derived feed may e.g. comprise a totalamount of free alpha-casein and free beta-casein in an amount in therange of 5-15% (w/w) relative to the total amount of protein of themilk-derived feed.

In some embodiments of the invention the milk-derived feed comprisesalpha-lactalbumin in an amount of at least 1% (w/w) relative to thetotal amount of protein of the milk-derived feed. For example, themilk-derived feed may comprise alpha-lactalbumin in an amount of atleast 10% (w/w) relative to the total amount of protein of themilk-derived feed, preferably at least 20% (w/w), and even morepreferably at least 30% (w/w) relative to the total amount of protein ofthe milk-derived feed.

In some embodiments of the invention the milk-derived feed comprisesalpha-lactalbumin in an amount in the range of 1-50% (w/w) relative tothe total amount of protein of the milk-derived feed. For example, themilk-derived feed may comprise alpha-lactalbumin in an amount in therange of 5-40% (w/w) relative to the total amount of protein of themilk-derived feed, preferably in the range of 10-35% (w/w), and evenmore preferably in the range of 12-30% (w/w) relative to the totalamount of protein of the milk-derived feed.

In some embodiments of the invention the milk-derived feed comprisesbeta-lactoglobulin in an amount of at least 1% (w/w) relative to thetotal amount of protein of the milk-derived feed. For example, themilk-derived feed may comprise beta-lactoglobulin in an amount of atleast 15% (w/w) relative to the total amount of protein of themilk-derived feed, preferably at least 30% (w/w), and even morepreferably at least 40% (w/w) relative to the total amount of protein ofthe milk-derived feed.

In some embodiments of the invention the milk-derived feed comprisesbeta-lactoglobulin in an amount in the range of 1-70% (w/w) relative tothe total amount of protein of the milk-derived feed. For example, themilk-derived feed may comprise beta-lactoglobulin in an amount in therange of 10-65% (w/w) relative to the total amount of protein of themilk-derived feed, preferably in the range of 20-60% (w/w), and evenmore preferably in the range of 35-55% (w/w) relative to the totalamount of protein of the milk-derived feed.

Caseins are known to precipitate at pH-values at or below about 4.6. Insome embodiments of the invention it is therefore preferred thatmilk-derived feed having a pH in the range of about 3.6-4.6 has arelatively low concentration of casein, such as at most 1% (w/w)relative to the total amount of protein of the milk-derived feed.

In some embodiments of the invention the milk-derived feed comprises atmost 0.1% casein (w/w) relative to the total amount of protein of themilk-derived feed. It may even be preferred that the milk-derived feedcomprises at most 0.01% casein (w/w) relative to the total amount ofprotein of the milk-derived feed.

In some preferred embodiments of the invention the milk-derived feedcomprises a total amount of protein in the range of 6-250 g/Lmilk-derived feed. For example, the milk-derived feed may comprise atotal amount of protein in the range of 50-150 g/L milk-derived feed. Itmay even be preferred that the milk-derived feed comprises a totalamount of protein in the range of 75-125 g/L milk-derived feed.

Alternatively, the milk-derived feed may comprise a total amount ofprotein in the range of 50-250 g/L milk-derived feed. For example, themilk-derived feed may comprise a total amount of protein in the range of50-150 g/L milk-derived feed. It may even be preferred that themilk-derived feed comprises a total amount of protein in the range of75-150 g/L milk-derived feed. It may also be preferred that themilk-derived feed may comprise a total amount of protein in the range of75-250 g/L milk-derived feed.

In some preferred embodiments of the invention the milk-derived feedcomprises a total amount of protein of at least 6 g/L milk-derived feed.For example, the milk-derived feed may comprise a total amount ofprotein of at least 50 g/L milk-derived feed. It may even be preferredthat the milk-derived feed comprises a total amount of protein of atleast 75 g/L milk-derived feed.

In some preferred embodiments of the invention the milk-derived feed hasa pH in the range of pH 3.8-6.0 at 25 degrees C. Preferably, the pH ofthe milk-derived feed at 25 degrees C. is in the range of pH 4.0-5.5.Even more preferably, the pH of the milk-derived feed at 25 degrees C.is in the range of pH 4.2-5.0, such as e.g. pH 4.3-4.5.

The desired specific conductance of the milk-derived feed may forexample be obtained by:

-   -   concentrating the milk-derived feed by partial removal of water        and/or addition of salt(s), which results in an increased        specific conductance, or    -   desalting the milk-derived feed by removal of salt(s) and/or        addition of water, which results in a reduced specific        conductance.

Techniques for removing water and/or salt(s) from an aqueous liquid arewell-known to the person skilled in the art.

In some preferred embodiments of the invention the milk-derived feed hasa specific conductance in the range of 4.5-9.0 mS/cm at 25 degrees C.For example, the specific conductance of the milk-derived feed may be inthe range of 5.0-8.0 mS/cm at 25 degrees C. In some preferredembodiments of the invention it may be even more preferable that thespecific conductance of the milk-derived feed is in the range of 5.5-7.0mS/cm at 25 degrees C.

The milk-derived feed may for example have a specific conductance in therange of 4-7 mS/cm at 25 degrees C. Alternatively, the milk-derived feedmay have a specific conductance in the range of 7-10 mS/cm at 25 degreesC. Alternatively, the milk-derived feed may have a specific conductancein the range of 5-8 mS/cm at 25 degrees C.

In some preferred embodiments of the invention the milk-derived feed hasa specific conductance in the range of 4-7 mS/cm at 25 degrees C. and apH in the range of pH 3.6-5.0 at 25 degrees C.

In other preferred embodiments of the invention the milk-derived feedhas a specific conductance in the range of 7-10 mS/cm at 25 degrees C.and a pH in the range of pH 5.0-6.5 at 25 degrees C.

In further preferred embodiments of the invention the milk-derived feedhas a specific conductance in the range of 5-8 mS/cm at 25 degrees C.and a pH in the range of pH 4.0-5.5 at 25 degrees C.

For example, the milk-derived feed may have a specific conductance inthe range of 5-6 mS/cm at 25 degrees C. and a pH in the range of pH4.2-5.0 at 25 degrees C.

In some embodiments of the invention, the milk-derived feed has a pH inthe range of 3.6 to 6.5 and

-   -   if the pH is in the range of 3.6-5.0, the specific conductance        is at least 4 mS/cm and at most cond._(min)=1.38 mS/cm*pH+1.03        mS/cm, and    -   if the pH is in the range of 5.0-6.5, the specific conductance        is at least cond._(min)=1.33 mS/cm *pH−2.67 mS/cm, and at most        cond._(max)=1.38 mS/cm*pH+1.03 mS/cm.

Thus, if the pH of a feed of these embodiments is e.g. pH 6.0 thespecific conductance is

-   -   at least cond._(min)=1.33 mS/cm*6.0-2.67 mS/cm=5.3 mS/cm, and    -   at most cond._(max)=1.38 mS/cm*6.0+1.03 mS/cm=9.3 mS/cm.

For example, the milk-derived feed may have a pH in the range of 3.6 to5.0 and a specific conductance of

-   -   at least 4 mS/cm and    -   at most cond._(max)=1.38 mS/cm*pH+1.03 mS/cm.

Alternatively, the milk-derived feed may have a pH in the range of 5.0to 6.5 and a specific conductance of

-   -   at least at least cond._(min)=1.33 mS/cm*pH−2.67 mS/cm and    -   at most cond._(max)=1.38 mS/cm*pH+1.03 mS/cm.

Specific conductivities and pH-values are measured in feeds having atemperature of 25 degrees C. unless it is stated otherwise.

In some embodiments of the invention the anion exchange medium comprisesa solid phase and one or more cationic groups.

Preferably, at least some of the cationic groups are attached to thesurface of the solid phase and/or to the surface of pores which areaccessible through the surface of the solid phase.

In some embodiments of the invention the solid phase of the anionexchange medium comprises one or more components selected from the groupconsisting of a plurality of particles, a filter, and a membrane.

The solid phase may for example comprise, or even essentially consistsof, polysaccharide.

Cross-linked polysaccharides are particularly preferred. Examples ofuseful polysaccharides are cellulose, agarose, and/or dextran.

Alternatively, the solid phase may comprise, or even essentiallyconsists of, a non-carbohydrate polymer. Examples of usefulnon-carbohydrate polymers are methacrylate, polystyrene, and/orstyrene-divinylbenzene.

In some preferred embodiments of the invention the cationic groupscomprises, or even essentially consists of, amino groups. Tertiary aminogroups are particularly preferred and result in quaternary ammoniumgroups under appropriate pH conditions. Quaternary ammonium groupsprovide strong anion exchange characteristics to the anion exchangemedium.

Alternatively, or additionally, the cationic groups may comprise one ormore primary or secondary amino groups. A substantial amount of primaryor secondary amino groups typically provides the anion exchange mediumwith weak anion exchange characteristics.

The optimal protein load per cycle depends on the design of the anionexchange chromatography system and the characteristics of the anionexchange medium.

The process conditions during the anion exchange chromatography,including pressure, flow rate, etc., depend on the actual processimplementation, the used equipment and the used anion exchange medium.

The temperature of the milk-derived feed during step b) is typicallysufficiently low to avoid microbial growth and heat damaging of theprotein and the anion exchange medium but sufficiently high to providean acceptable viscosity.

In some embodiments of the invention the temperature of the milk-derivedfeed during step b) is in the range of 2-40 degrees C. Preferably, thetemperature of the milk-derived feed during step b) is in the range of4-20 degrees C., and even more preferably in the range of 6-12 degreesC. More details regarding anion exchange chromatography and itsindustrial implementation can be found in Scopes, which is incorporatedherein by reference for all purposes.

In some preferred embodiments of the invention the method of theinvention comprises a step c) of washing the anion exchange medium witha washing solution after contacting it with the milk-derived feed.Useful washing solutions are typically pH neutral or weak acidic aqueoussolutions capable of removing loosely bound molecules from the anionexchange medium. One may for example use demineralised water or a pHneutral aqueous solution of sodium chloride, e.g. 0.1 M NaCl, as washingliquid.

In other preferred embodiments of the invention, the method of thepresent invention does not contain step c).

Step d) of the present invention involves recovering the osteopontinbound to the anion exchange medium. The recovery is typically performedby contacting the anion exchange medium with an eluent and collectingthe resulting eluate, i.e. the eluent plus the molecules released fromthe anion exchange medium.

Normally, the eluent is an aqueous solution having an ion strengthand/or pH sufficient to release bound osteopontin from the anionexchange medium. Examples of useful eluents are pH-neutral, 1.0 Maqueous solutions of salts such as NaCl, CaCl₂, KCl, MgCl₂, or acombination thereof.

The recovered composition may be subjected to additional process stepse.g. for demineralising and concentrating the composition, andsubsequently transforming it into a powder.

Thus, in some preferred embodiments of the invention, the recoveredcomposition is furthermore subjected to one or more of the processstep(s) selected from the group consisting of concentration,diafiltration, evaporation of solvent, spray-drying, and substitution ofprotein-bound cations.

For example, the recovered composition may be subjected to aconcentration step.

Alternatively, or in addition, the recovered composition may besubjected to a diafiltration step.

Alternatively, or in addition, the recovered composition may besubjected to an evaporation step.

Alternatively, or in addition, the recovered composition may besubjected to a spray-drying step.

In a preferred embodiment of the invention the recovered composition issubjected to the following steps:

-   -   i) concentrating, e.g. by ultrafiltration,    -   ii) diafiltration, e.g. against water,    -   iii) optionally, another concentration step, e.g. by        evaporation,    -   iv) cation replacement by contacting the aqueous composition of        step ii) or iii) with a water soluble calcium salt, e.g. CaCl₂,    -   v) pasteurisation, and    -   vi) spray-drying to convert the pasteurised composition into a        powder.

The present method may both be implemented as a batch process or asemi-batch-process. The semi-batch process may for example beimplemented by operating a first and a second anion exchange column, andperforming step b) on the first anion exchange column while performingsteps c) and/or d) on the second anion exchange column, and vice versa.

In some embodiments of the invention the present method for isolatingosteopontin from a milk-derived feed comprises the steps of:

-   -   a) providing a milk-derived feed comprising osteopontin, said        milk-derived feedhaving a pH in the range of pH 3.6-6.5 at 25        degrees C. and a specific conductance in the range of 4-10 mS/cm        at 25 degrees C., and wherein the milk-derived feed comprises a        total amount of protein in the range of 50-250 g/L milk-derived        feed, and wherein the milk-derived feed furthermore comprises        additional protein having an isoelectric point (pI) <5.0.    -   b) subjecting said milk-derived feed to anion exchange        chromatography, which includes contacting the milk-derived feed        with an anion exchange medium,    -   c) optionally, washing the anion exchange medium, and    -   d) recovering the protein bound to the anion exchange medium,        thereby obtaining a composition comprising isolated osteopontin.

In other embodiments of the invention the present method for isolatingosteopontin from a milk-derived feed comprises the steps of:

-   -   a) providing a milk-derived feed comprising osteopontin, said        milk-derived feed having a the milk-derived feed has a pH in the        range of 3.6 to 6.5 and        -   if the pH is in the range of 3.6-5.0, the specific            conductance is at least 4 mS/cm and at most cond._(max)=1.38            mS/cm*pH+1.03 mS/cm, and        -   if the pH is in the range of 5.0-6.5, the specific            conductance is        -   at least cond._(min)=1.33 mS/cm *pH−2.67 mS/cm, and        -   at most cond._(max)=1.38 mS/cm*pH+1.03 mS/cm, and        -   wherein milk-derived feed comprises a total amount of            protein in the range of 50-250 g/L milk-derived feed,    -   b) subjecting said milk-derived feed to anion exchange        chromatography, which includes contacting the milk-derived feed        with an anion exchange medium,    -   c) optionally, washing the anion exchange medium, and    -   d) recovering the protein bound to the anion exchange medium,        thereby obtaining a composition comprising isolated osteopontin.

In further embodiments of the invention the present method for isolatingosteopontin from a milk-derived feed comprises the steps of:

-   -   a) providing a milk-derived feed comprising osteopontin, said        milk-derived feed having a the milk-derived feed has a pH in the        range of 3.6 to 6.5 and        -   if the pH is in the range of 3.6-5.0, the specific            conductance is at least 4 mS/cm and at most cond._(min)=1.38            mS/cm*pH+1.03 mS/cm, and        -   if the pH is in the range of 5.0-6.5, the specific            conductance is at least cond._(min)=1.33 mS/cm *pH−2.67            mS/cm, and        -   at most cond._(max)=1.38 mS/cm*pH+1.03 mS/cm, and        -   wherein milk-derived feed comprises a total amount of            protein in the range of 50-250 g/L milk-derived feed, and            wherein the milk-derived feed furthermore comprises            additional protein having an isoelectric point (pI) <5.0,    -   b) subjecting said milk-derived feed to anion exchange        chromatography, which includes contacting the milk-derived feed        with an anion exchange medium,    -   c) optionally, washing the anion exchange medium, and    -   d) recovering the protein bound to the anion exchange medium,        thereby obtaining a composition comprising isolated osteopontin.

Yet an aspect of the invention relates to an osteopontin-containingcomposition obtainable by the method described herein.

The present invention has been described above with reference tospecific embodiments. However, other embodiments than the abovedescribed are equally possible within the scope of the invention. Thedifferent features and steps of various embodiments and aspects of theinvention may be combined in other ways than those described hereinunless it is stated otherwise.

EXAMPLES Example 1 Enrichment of Osteopontin from Controlled ConductanceSweet Whey Concentrate

Protocol

The following purification experiments were conducted on Fast ProteinLiquid Chromatography (FPLC) equipment equipped with the strong anionexchange resin Q-Sepharose Bigbeads packed in a 13.3 mL (170 mm×10 mm)column. The flow was maintained at 3.33 ml/min throughout the loading,wash and elution in all experiments. The raw material was a sweet wheyprotein isolate and 70 g of total protein was loaded on the columnduring each experiment. The content of CMP in the raw material wasanalysed. Samples of the raw material was diluted to a final proteinconcentration of 10% w/v, hereafter pH (pH 3.0-5.7, at 4.7 mS/cm) andspecific conductance (2.3-10.3 mS/cm at pH 4.3) were adjusted byaddition of HCl and NaCl, respectively. Unbound proteins were washed outof the column by 3 bed volumes of 100 mM NaCl, pH 5.0 solution.Subsequently, the bound proteins were eluted by passage of 1 M NaCl, pH5.0 solution through the column until no more protein was detected inthe eluate. For each eluate sample, the contents of osteopontin (OPN),CMP, and total protein were analysed using the following techniques.

Determination of Total Protein

Total protein content of the eluate was measured by standard Kjeldahldigestion as described in Cohen.

OPN Quantification by HPLC Method

Analytical Principle:

The sample was filtered through a 0.22 μm filter and subjected to HPLCwith column MonoQ HR 5/5 (1 ml), Pharmacia and detection at 280 nm. Theconcentration of the sample was calculated by the external standardmethod (comparison with peak area of standard with known OPN content).It is a prerequisite for this analytical procedure that the samplescomprise relatively pure OPN, e.g. as determined by SDS-PAGE (Laemmli,1970), due to the low specificity of the method.

Reagents: OPN standard, Milli Q water, HPLC grade, NaCl, Merck, TrisHCl, Sigma

Buffer A: 10 mM NaCl, 20 mM Tris HCL, pH 8.0

Buffer B: 0.8 M NaCl, 20 mM Tris HCl, pH 8.0

A standard calibration curve was made from 5 standards in theconcentration range 1-10 mg/ml of OPN standard in buffer A. Allstandards were filtered by 0.22 μm filters before loading onto thecolumn.

Sampling and Pretreatment:

Samples for analysis were diluted with Milli Q water, HPLC grade, ifthey were out of range of the standard calibration curve. Dilution wasin some instances also necessary to enable binding of OPN to the anionexchange resin if much NaCl from the eluent was present. An amountequivalent to 25 μL of 1-10 mg/mL OPN was injected for analysis. Sampleswere filtered through 0.22 μm filters before injection to HPLC.

HPLC conditions: Flow 1 ml/min, injection volume 25 μL, gradient: 0-3min 0% B, 3-17 min 0-60% B, 17-30 min 60-100% B, 30-33 min 100% B, 33-34min 100-0% B, 34-40 min 0% B.

Calculation and Expression of results:

The concentration of OPN in each sample was calculated by reference tothe standard curve and by observing the employed dilutions.

The purity was calculated by the ratio between OPN and total protein,using the OPN specific Jones factor of 7.17 as most or all proteindetermined by Kjeldahl digestion in the current context was OPN.

Quantification of Major Whey Proteins by HPLC

Separation and quantification of major whey protein, alpha-lactalbumin,β-lactoglobulin and CMP (caseino glycomacropeptide) by gel permeationchromatography.

The gel permeation chromatography was conducted using 2 columns ofTSKge13000PWx1 (7.8 mm×30 cm) connected in series with attachedprecolumn PWx1 (6 mm 4 cm)(Tosohass, Japan). The standard solution forcalibration of the system consisted of: 225 mg CMP; 225 mgalpha-lactalbumin and 50 mg β-lactoglobulin dissolved in 500.0 ml 0.02 Mphosphate buffer 7.5. Pretreatment of samples: Powder samples weredissolved in phosphate buffer at 1 mg/ml and left over night forsolubilisation. Alternatively, liquid samples were diluted withphosphate buffer to obtain a content of approx. 0.1% protein. If theprotein content was below 0.1%, the sample was measured undiluted. Allsamples were filtered through 0.22 μm filters before injection to thecolumn.

Calculation and Expression of results:

Percentage of individual protein in sample was calculated according toA×0.1×F, where

-   -   B

A=area measured in sample

0.1=conversion factor for 0.1% solution

F=dilution factor (100,000/sample weight in mg)

B=calculated area of individual protein in 0.1% standard solution

Results

Content of CMP

The content of CMP in the sweet whey raw material was 20.1%, but CMP wassurprisingly undetectable in the enriched OPN compositions of Example 1.

The effect of feed pH on purity and yield of osteopontin:

The first series of experiments with varying the pH value of the appliedfeed, shown in FIG. 1, show that the yield of OPN (mg OPN from 70 gtotal protein in the feed) increases as the pH increases from pH 3 toapprox. pH 4.3. The high yield is maintained when the pH is increasedbeyond pH 4.3. The purity of OPN in the obtained OPN preparations isvery high at low pH values, but when the pH is increased above approx.pH 4.5 the purity slowly declines but is still acceptable until approx.pH 6.5. However, the best yield of high purity OPN is obtained in the pHwindow pH 3.8-5.5, and particularly in the range pH 4.3-4.5.

The effect of specific conductance of the feed on purity and yield ofosteopontin:

The results from the series of experiments with varying specificconductance are shown in FIG. 2. At low specific conductance the yieldis high while the purity is low. With increasing specific conductancethe purity increases and reaches 100% at approx. 5.5 mS/cm. When thespecific conductance is increased further the purity remains high,whereas the yield of OPN starts to decline when the specific conductanceexceeds approx. 10 mS/cm. However, the best yield of high purity OPNseems to obtained in the specific conductance range 4.5-9.0 mS/cm, andparticularly in the range 5-8 mS/cm.

Discussion/Conclusion

Effect of pH:

It is seen from FIG. 1 that at a pH value below approx. pH 3.6 OPN isdecreasing its charge and hence it is decreasing its binding to thecationic groups of the resin. At pH values above approx. pH 4.5 thepurity of OPN starts to decrease as other proteins of the feed becomenegatively charged and start binding to the resin. We have thusidentified a window of optimal pH values in the range pH 3.6-6.5 forisolating OPN of high purity and with high yield even though the feedcontains a large amount of CMP. In the resulting OPN preparation CMP isnot detectable.

Effect of specific conductance:

It is seen from FIG. 2 that at a specific conductance below approx. 4mS/cm the purity of OPN is decreasing as other proteins can bind to theresin. At specific conductances above approx. 6 mS/cm the binding of OPNto the resin becomes weaker and the yield slowly decreases but isacceptable until a specific conductance of approx. 10 mS/cm. Thus, wehave identified a narrow range from approx. 4-10 mS/cm to be optimal forOPN production. In this narrow range OPN can be isolated in high yieldand with high purity even from a raw material which contains approx. 20%CMP.

Several additional experiments have been performed exploring the claimedpH/specific conductance window in sweet whey based-feeds and theadditional experiments confirm the above-mentioned conclusions.

Example 2 Enrichment of Osteopontin from Controlled Conductance AcidWhey Concentrate

The following purification experiment was conducted on FPLC equipmentsimilar to Example 1. The raw material, however, was acid wheyconcentrate having a total protein content of 10% (w/w) and approx. 70 gof total protein was loaded on the column.

The concentrated acid whey was diluted to different degrees giving riseto specific conductivities in the range from 2.5 to 10 mS/cm. OPN andtotal protein was measured in the feed and the eluate by the methodsdescribed in Example 1.

The pH was adjusted to 4.3 by addition of HCl and the raw materialapplied to the anion exchange column. Unbound proteins were washed outof the column by 3 bed volumes of 0.1 M NaCl and the bound proteins weresubsequently eluted by passage of 1 M NaCl solution through the columnuntil no more protein was detected in the eluate.

The influence of the specific conductance on OPN purity and yield duringOPN enrichment from concentrated acid whey is shown in FIG. 3.

The data in FIG. 3 shows an enrichment of OPN from acid whey by thedescribed procedure.

As in Example 1 where sweet whey was used as raw material, it is againobserved that low specific conductance in the feed increases the yieldon the sacrifice of purity of the product.

However, as acid whey derived feeds do not contain large quantities ofCMP the effect is not as detrimental to the enrichment of OPN as in thecase where the feed is derived from sweet whey. Acid whey derived feeddoes contain minor components, which binding can be minimised by thesame parameter settings as employed to avoid binding of CMP. Thus, itcan be observed that the narrow optimum range of OPN isolationidentified in Example 1 applies to the OPN isolation from acid whey aswell.

We have performed several additional experiments exploring the claimedpH/specific conductance window in acid whey based-feeds and theadditional experiments confirm the above-mentioned findings.

Example 3 Enrichment of Osteopontin from Controlled Conductance MilkSerum Concentrate

In accordance with the previous examples milk serum can be used as rawmaterial for OPN production by the disclosed method. Milk serum wasproduced by microfiltration (filter pore size of approx. 0.1 micron) ofskimmed milk at 24 degrees so as to remove micellar casein. Samples ofmilk serum were thereafter adjusted to pH 4.3 or pH 4.7 by HCl anddiafiltered to obtain a specific conductance in the range of 4-10 mS/cmand a total protein content of approx. 5% or 10% (w/w). Subsequently,OPN was enriched from the modified milk serum samples by anion exchangeat approx. 5 degrees C. as described in example 1.

Results and Observations

The performed trials demonstrated that feeds derived from milk serumalso may be used in the present process.

Surprisingly, unidentified precipitation was observed during the processresulting in clogging or contamination of the anion exchange materialafter some cycles of anion exchange. The precipitation problem wassolved by filtering the acidified milk serum through filter paper priorto the anion exchange. We have seen indications that the precipitate isrelated to dissolved casein species, such as free alpha- and freebeta-casein that stay in the milk serum when the native casein micellesare removed.

Discussion/Conclusion

Milk serum is produced by microfiltration of skimmed milk so as toremove caseins, whereas sweet and acid whey are casein depleted byprecipitation of caseins by action of rennet or acid, respectively.Thereby all three categories of milk derived feeds are essentially freeof native casein micelles, which would interfere with anion exchangechromatographic procedures due to precipitation and flocculation ofcaseins.

We have seen indications that dissolved caseins cause problems duringanion exchange. This problem can solved by filtering the milk derivedfeeds prior to anion exchange using e.g. a microfilter.

Alternatively, the anion exchange step may be performed at a pH whereprecipitation is limited or even absent, e.g. in the range of pH5.0-6.5. When performing the anion exchange in this pH range it isfurthermore suggested to use a feed/process temperature above 15 degreesC., such as 20-40 degrees C. In this temperature range dissolvedbeta-casein forms small beta-casein micelles, not to be confused withnative casein micelles, and these beta-casein micelles interfere lesswith the anion exchange process than single beta-casein molecules.

Example 4 Enrichment of Osteopontin from Sweet Whey Concentrate withoutControlled Conductance

The following purification experiment was conducted on FPLC equipmentsimilar to Example 1. The raw material was sweet whey isolate andapprox. 1 g of total protein was loaded on the column. Before loading tothe column the sweet whey was concentrated by ultrafiltration,diafiltered after addition of an equal volume of water and finallydiluted again by an equal volume of water. The resulting specificconductance was 2.1 mS/cm. CMP and total protein was measured in feedand eluate by the methods described in Example 1. The amount of OPNcould not be measured due to interfering proteins in both feed andeluate. OPN data in Table 1 is estimated from known yields inexperiments from Example 1.

The pH was adjusted to 4.3 by addition of HCl and the raw materialapplied to the anion exchange column. Unbound proteins were washed outof the column by 3 bed volumes of water and the bound proteins werehereafter eluted by passage of 1 M NaCl solution through the columnuntil no more protein was detected in the eluate.

TABLE 1 Results of anion exchange of low conductance sweet whey Rawmaterial Eluate Total protein, mg 979 192 OPN, mg 2.9 2.8 Purity of OPN,% 0.3 1.5 GMP, mg 193 186 Purity GMP, % 19.7 97Discussion/Conclusion:

The data in Table 1 show an enrichment of acidic whey proteins by thedescribed procedure. However, OPN is only enriched to a minor extendbecause it constitutes a minor fraction of acidic whey proteins in thefeed. As e.g. CMP is also binding to the resin and is present in thefeed in a high concentration this protein takes up a large amount of thebinding capacity of the resin. Therefore this procedure is notsatisfactory for OPN production.

Example 5 Comparison and Conclusion (Comparing Example 1 with Example 4)

Some characteristics of the processes in Examples 1 and 4 are comparedin Table 2 below.

TABLE 2 Characteristics of Example 1 and 4 Example 1 Example 4 pH 4.34.3 Specific conductance, mS/cm 6.0 2.1 Protein load pr. ml resin, g/ml5.26 0.074 Estimated OPN of protein in raw material, % 0.3 0.3 Foldenrichment of OPN 333 5 Purity of OPN in eluate, % 100 1.5Conclusion

As summarized in Table 2, the method of Example 1 is much more specificfor enriching OPN than the method of Example 4. Thus, much more proteincan be loaded to the column per cycle and the yield of OPN per cycle ismuch higher with the method of Example 1. This method results inpractically pure OPN, whereas CMP dominates the product of Example 4.

References

-   Cohen Julius B. Cohen, Practical Organic Chemistry, 1910-   Evans et al. Evans et al. , “Comparison of composition, sensory, and    volatile components of thirty-four percent whey protein and milk    serum protein concentrates”, J. Dairy Sci. 92:4773-4791, 2009-   Scopes Protein Purification: Principles and Practice; Robert K.    Scopes; 3rd edition, Springer Verlag New York, Inc., ISBN    0-387-94072-3-   WO 02/28413 A 1-   WO 01/149741 A2-   WO 99/33415 A1

The invention claimed is:
 1. A method for isolating osteopontin from amilk-derived feed, the method comprising the steps of: a) providing amilk-derived feed comprising osteopontin, said milk-derived feed havinga pH in the range of pH 3.6-6.5 at 25 degrees C. and a specificconductance in the range of 4-10 mS/cm at 25 degrees C., and wherein themilk-derived feed comprises a total amount of protein in the range of50-250 g/L milk-derived feed, b) subjecting said milk-derived feed toanion exchange chromatography, which includes contacting themilk-derived feed with an anion exchange medium, c) optionally, washingthe anion exchange medium, and d) recovering the protein bound to theanion exchange medium, thereby obtaining a composition comprisingisolated osteopontin.
 2. The method according to claim 1, wherein themilk-derived feed furthermore comprises additional protein having anisoelectric point (pI) <5.0.
 3. The method according to claim 2, whereinthe one or more additional protein(s) comprises one or more protein(s)selected from the group consisting of alpha lactalbumin, caseinomacropeptide, caseino phosphopeptide, proteose peptone-3, proteosepeptone-5, proteose peptone-8, and a mixture thereof.
 4. The methodaccording to claim 3, wherein the one or more additional protein(s)comprises caseino macropeptide (CMP).
 5. The method according to claim1, wherein the milk-derived feed comprises a total amount of protein inthe range of 50-150 g/L milk-derived feed.
 6. The method according toclaim 1, wherein the milk-derived feed comprises a total amount ofprotein in the range of 75-125 g/L milk-derived feed.
 7. The methodaccording to claim 1, wherein the milk-derived feed comprises a milkserum-derived feed.
 8. The method according to claim 1, wherein themilk-derived feed comprises sweet whey-derived feed.
 9. The methodaccording to claim 1, wherein the milk-derived feed comprises CMP in anamount of at least 1% (w/w) relative to the total amount of protein ofthe milk-derived feed.
 10. The method according to claim 1, wherein themilk-derived feed comprises CMP in an amount in the range of 1-40% (w/w)relative to the total amount of protein of the milk-derived feed. 11.The method according to claim 1, wherein the milk-derived feed comprisesalpha-lactalbumin in an amount of at least 1% (w/w) relative to thetotal amount of protein of the milk-derived feed.
 12. The methodaccording to claim 1, wherein the milk-derived feed comprisesalpha-lactalbumin in an amount in the range of 1-50% (w/w) relative tothe total amount of protein of the milk-derived feed.
 13. The methodaccording to claim 1, wherein the milk-derived feed comprisesbeta-lactoglobulin in an amount of at least 1% (w/w) relative to thetotal amount of protein of the milk-derived feed.
 14. The methodaccording to claim 1, wherein the milk-derived feed comprisesbeta-lactoglobulin in an amount in the range of 1-70% (w/w) relative tothe total amount of protein of the milk-derived feed.
 15. The methodaccording to claim 1, wherein the milk-derived feed has a pH in therange of pH 3.8-5.5 at 25 degrees C.
 16. The method according to claim1, wherein the milk-derived feed has a specific conductance in the rangeof 4.5-9.0 mS/cm at 25 degrees C.
 17. The method according to claim 1,wherein the milk-derived feed has a specific conductance in the range of4-7 mS/cm at 25 degrees C.
 18. The method according to claim 1, whereinthe milk-derived feed has a specific conductance in the range of 5-8mS/cm at 25 degrees C.
 19. The method according to claim 1, wherein themilk-derived feed has a specific conductance in the range of 7-10 mS/cmat 25 degrees C.
 20. The method according to claim 1, wherein the anionexchange medium comprises a solid phase and one or more cationic groups.21. The method according to claim 20, wherein the solid phase of theanion exchange medium comprises one or more components selected from thegroup consisting of a plurality of particles, a filter, and a membrane.22. The method according to claim 20, wherein the solid phase comprisespolysaccharide.
 23. The method according to claim 20, wherein the solidphase comprises cross-linked polysaccharide.
 24. The method according toclaim 20, wherein the solid phase comprises sepharose.
 25. The methodaccording to claim 20, wherein the cationic groups comprises aminogroups.
 26. The method according to claim 1, wherein the recoveredcomposition furthermore is subjected to one or more of the processstep(s) selected from the group consisting of concentration,diafiltration, evaporation of solvent, spray-drying, and substitution ofprotein-bound cations.
 27. The method according to claim 1, wherein theisolated osteopontin comprises at least 70% (w/w) relative to totalweight of protein recovered from the anion exchange medium.